It is proposed to determine the stoichiometric and thermodynamic parameters of the interactions between tubulin and ligands which affect the structural integrity of microtubules. The ultimated goal of this investigation is to elucidate the mechanisms of regulating the assembly of microtubule and the role of microtubule events. This research proposal is initiated as a result of recent reports on the important role of a variety of small molecules in affecting the structural integrity of microtubule. To achieve such an objective, the specific aim of this research program is to study the interaction between tubulin and these ligands such as cyclic nucleotides, monovalent cation, an anticancer agent and anesthetics. The stoichiometry and thermodynamic parameters governing the interactions between tubulin and these ligands will be determined by the method of gel filtration. Since the effects of ligands may be a manifestation of an alteration in tubulin interaction with factors essential for microtubule assembly, it is proposed to study the binding of ligands as a function of Mg ions and GTP concentrations. The free energy coupling between ligands can then be determined, quantitating the co-operativity among ligands. The effects of ligands on tubulin self-association to structures other than microtubule will be studied by sedimentation velocity with the aim of obtaining thermodynamic parameters characterizing these reactions and the hydrodynamic properties of the aggregates formed. The self-assembly of tubulin to microtubules will be monitored by turbidity measurements and electron microscopy.